Within the past four years, the genetic cause of the Hereditary Nonpolyposis Colorectal Cancer Syndrome (HNPCC), also known as Lynch syndrome II, has been ascertained for the majority of kindreds affected with the disease (13). The molecular basis of HNPCC involves genetic instability resulting from defective mismatch repair (MMR). Many genes have been identified in rodents and humans that encode for proteins that appear to participate in the MMR process, including the mutS homologs GTBP, hMSH2, and hMSH3 and the mutL homologs hMLH1, hPMS1, and hPMS2 (2, 7, 11, 17, 20, 21, 22, 24). Germ line mutations in four of these genes (hMSH2, hMLH1, hPMS1, and hPMS2) have been identified in HNPCC kindreds (2, 11, 13, 17, 24). Though the mutator defect that arises from the MMR deficiency can affect any DNA sequence, microsattelite sequences are particularly sensitive to MMR abnormalities (14). Microsattelite instability is therefore a useful indicator of defective MMR. In addition to its occurrence in virtually all tumors arising in HNPCC patients, Microsattelite instability is found in a small fraction of sporadic tumors with distinctive molecular and phenotypic properties (27).
HNPCC is inherited in an autosomal dominant fashion, so that the normal cells of affected family members contain one mutant allele of the relevant MMR gene (inherited from an affected parent) and one wildtype allele (inherited from the unaffected parent). During the early stages of tumor development, however, the wildtype allele is inactivated through a somatic mutation, leaving the cell with no functional MMR gene and resulting in a profound defect in MMR activity. Because a somatic mutation in addition to a germline mutation is required to generate defective MMR in the tumor cells, this mechanism is generally referred to as one involving two hits, analogous to the biallelic inactivation of tumor suppressor genes that initiate other hereditary cancers (11, 13, 25). In line with this two hit mechanism, the non-neoplastic cells of HNPCC patients generally retain near normal levels of MMR activity due to the presence of the wildtype allele.
A wide range of organisms with defective MMR have been found to have widespread genetic mutations throughout their genome. In all cases, these organisms have germline mutations within both copies of a particular MMR gene. Recently, work done by Nicolaides et al have shown that a decrease in MMR can be achieved within cells from higher order organisms by introducing a dominant negative allele of a MMR gene. These data suggest that the use of such an approach can generate genetically altered organisms to produce new output traits. There is a need in the art for additional methods with which to generate genetic diversity.